Investigation of the mechanism of action of hexamethylene bisacetamide (HMBA), a potent inducer of terminal erythroid differentiation in murine erythroleukemia cells (MELC), will be pursued. Culture of MELC in the presence of 5mM HMBA induces essentially all cells (less than 99%) to differentiate. Morphological changes characteristic of erythroid development and accumulation of globin mRNA and hemoglobin occur. Differentiating cells are limited in their proliferative capacity to 5 or fewer divisions. MELC cultured in the presence of HMBA for 24 hours exhibit commitment to differentiate in that they continue development in the absence of inducer. Studies will focus on events occurring during the first 24 hours of culture. Cellular uptake, metabolism, and intracellular distribution of HMBA and its metabolic products will be studied employing HMBA radioactively labeled in various portions of the molecule. Cell fractionation techniques suitable for MELC will be determined and purified components analyzed for content of HMBA and its metabolic products. Studies will involve characterization of the earliest effect of HMBA and correlation with commitment to differentiate. The relationship between molecular structure and inducing activity will be investigated by synthesizing compounds chemically related to HMBA and testing for their ability to induce differentiation.